科微学术

生物工程学报

2025年4月2日 8:07 星期三
首页 > 过刊浏览>2023年第39卷第6期 >2215-2230. DOI:10.13345/j.cjb.230111
PDF HTML阅读 XML下载 导出引用 引用提醒
功能膜微域在七烯甲萘醌合成过程中的作用解析
作者:
基金项目:

国家自然科学基金(22278186， 22208122)


Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7
Author:
  • 摘要
    • | |
  • 访问统计
    • |
  • 参考文献 [34]
    • |
  • 相似文献 [20]
    • | | |
  • 文章评论
    摘要:

    功能膜微域(functional membrane microdomains, FMMs)是细菌细胞质膜上富含脚手架蛋白和聚异戊二烯类物质的结构域，参与细胞生命活动的多个过程。本研究主要聚焦于揭示FMMs与MK-7之间的相关性，并对MK-7合成进行代谢调控。首先，通过荧光标记初步确定FMMs和MK-7在细胞膜上存在相关性；其次通过分析FMMs破坏前后细胞膜上MK-7含量的变化以及细胞膜有序度的变化情况，明确MK-7是FMMs的聚异戊二烯类关键组分；接下来，采用可视化分析探究MK-7合成过程中部分关键酶的亚细胞定位，并通过FloA将胞内游离的途径酶Fni、IspA、HepT和YuxO定位至FMMs中，进而实现MK-7合成途径的区室化，最终成功获得一株高产MK-7的枯草芽孢杆菌(Bacillus subtilis)菌株BS3AT，摇瓶水平MK-7产量达到300.3 mg/L，3 L发酵罐中MK-7产量为464.2 mg/L。

    Abstract:

    Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

    参考文献
    [1] GRÖBER U, REICHRATH J, HOLICK MF, KISTERS K. Vitamin K: an old vitamin in a new perspective[J]. Dermato-Endocrinology, 2014, 6(1): e968490.
    [2] BEULENS JWJ, BOOTH SL, van den HEUVEL EGHM, STOECKLIN E, BAKA A, VERMEER C. The role of menaquinones (vitamin K2) in human health[J]. British Journal of Nutrition, 2013, 110(8): 1357-1368.
    [3] DALMEIJER GW, van den SCHOUW YT, MAGDELEYNS E, AHMED N, VERMEER C, BEULENS JWJ. The effect of menaquinone-7 supplementation on circulating species of matrix Gla protein[J]. Atherosclerosis, 2012, 225(2): 397-402.
    [4] PAINTER KL, HALL A, HA KP, EDWARDS AM. The electron transport chain sensitizes Staphylococcus aureus and Enterococcus faecalis to the oxidative burst[J]. Infection and Immunity, 2017, 85(12): e00659-17.
    [5] YANG SM, CAO YX, SUN LM, LI CF, LIN X, CAI ZG, ZHANG GY, SONG H. Modular pathway engineering of Bacillus subtilis to promote de novo biosynthesis of menaquinone-7[J]. ACS Synthetic Biology, 2019, 8(1): 70-81.
    [6] MEGANATHAN R. Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms[J]. Vitamins & Hormones, 2001, 61: 173-218.
    [7] JOHNSTON JM, BULLOCH EM. Advances in menaquinone biosynthesis: sublocalisation and allosteric regulation[J]. Current Opinion in Structural Biology, 2020, 65: 33-41.
    [8] HIROTA Y, NAKAGAWA K, SAWADA N, OKUDA N, SUHARA Y, UCHINO Y, KIMOTO T, FUNAHASHI N, KAMAO MY, TSUGAWA N, OKANO T. Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1[J]. Public Library of Science, 2015, 10(4): e0125737.
    [9] LIU Y, YANG ZM, XUE ZL, QIAN SH, WANG Z, HU LX, WANG J, ZHU H, DING XM, YU F. Influence of site-directed mutagenesis of UbiA, overexpression of dxr, menA and ubiE, and supplementation with precursors on menaquinone production in Elizabethkingia meningoseptica[J]. Process Biochemistry, 2018, 68: 64-72.
    [10] 崔世修. 代谢工程改造枯草芽孢杆菌高效合成七烯甲萘醌[D]. 无锡: 江南大学博士学位论文, 2020. CUI SX. Metabolic engineering of Bacillus subtilis for efficient synthesis of menaquinone-7[D]. Wuxi: Doctoral Dissertation of Jiangnan University, 2020 (in Chinese).
    [11] MA YW, MCCLURE DD, SOMERVILLE MV, PROSCHOGO NW, DEHGHANI F, KAVANAGH JM, COLEMAN NV. Metabolic engineering of the MEP pathway in Bacillus subtilis for increased biosynthesis of menaquinone-7[J]. ACS Synthetic Biology, 2019, 8(7): 1620-1630.
    [12] CUI SX, LV XQ, WU YK, LI JH, DU GC, LEDESMA-AMARO R, LIU L. Engineering a bifunctional Phr60-Rap60-Spo0A quorum-sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis[J]. ACS Synthetic Biology, 2019, 8(8): 1826-1837.
    [13] FARHI M, MARHEVKA E, MASCI T, MARCOS E, EYAL Y, OVADIS M, ABELIOVICH H, VAINSTEIN A. Harnessing yeast subcellular compartments for the production of plant terpenoids[J]. Metabolic Engineering, 2011, 13(5): 474-481.
    [14] LAUDE AJ, PRIOR IA. Plasma membrane microdomains: organization, function and trafficking[J]. Molecular Membrane Biology, 2004, 21(3): 193-205.
    [15] YEE DA, DENICOLA AB, BILLINGSLEY JM, CRESO JG, SUBRAHMANYAM V, TANG Y. Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2019, 55: 76-84.
    [16] SHENG JY, STEVENS J, FENG XY. Pathway compartmentalization in peroxisome of Saccharomyces cerevisiae to produce versatile medium chain fatty alcohols[J]. Scientific Reports, 2016, 6: 26884.
    [17] DELOACHE WC, RUSS ZN, DUEBER JE. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways[J]. Nature Communications, 2016, 7: 11152.
    [18] AVALOS JL, FINK GR, STEPHANOPOULOS G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols[J]. Nature Biotechnology, 2013, 31(4): 335-341.
    [19] BRAMKAMP M, LOPEZ D. Exploring the existence of lipid rafts in bacteria[J]. Microbiology and Molecular Biology Reviews, 2015, 79(1): 81-100.
    [20] PIKE LJ. Lipid rafts: bringing order to chaos[J]. Journal of Lipid Research, 2003, 44(4): 655-667.
    [21] OTTO GP, NICHOLS BJ. The roles of flotillin microdomains-endocytosis and beyond[J]. Journal of Cell Science, 2011, 124(23): 3933-3940.
    [22] STUERMER CAO. Reggie/flotillin and the targeted delivery of cargo[J]. Journal of Neurochemistry, 2011, 116(5): 708-713.
    [23] LV XQ, ZHANG C, CUI SX, XU XH, WANG LL, LI JH, DU GC, CHEN J, LEDESMA-AMARO R, LIU L. Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis[J]. Metabolic Engineering, 2020, 61: 96-105.
    [24] OWEN DM, MAGENAU A, WILLIAMSON D, GAUS K. The lipid raft hypothesis revisited-new insights on raft composition and function from super-resolution fluorescence microscopy[J]. BioEssays, 2012, 34(9): 739-747.
    [25] LANGHORST MF, REUTER A, STUERMER CAO. Scaffolding microdomains and beyond: the function of reggie/flotillin proteins[J]. Cellular and Molecular Life Sciences CMLS, 2005, 62(19/20): 2228-2240.
    [26] CHAZOTTE B. Labeling mitochondria with rhodamine 123[J]. Cold Spring Harbor Protocols, 2011, 2011(7): pdb.prot5640.
    [27] FENG XX, HU YM, ZHENG YY, ZHU W, LI K, HUANG CH, KO TP, REN FF, CHAN HC, NEGA M, BOGUE S, LÓPEZ D, KOLTER R, GÖTZ F, GUO RT, OLDFIELD E. Structural and functional analysis of Bacillus subtilis YisP reveals a role of its product in biofilm production[J]. Chemistry & Biology, 2014, 21(11): 1557-1563.
    [28] BOSAK T, LOSICK RM, PEARSON A. A polycyclic terpenoid that alleviates oxidative stress[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(18): 6725-6729.
    [29] DINIC J, BIVERSTÅHL H, MÄLER L, PARMRYD I. Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2011, 1808(1): 298-306.
    [30] JIN L, LI LH, ZHANG WC, ZHANG RZ, XU Y. Heterologous expression of bovine lactoferrin C-lobe in Bacillus subtilis and comparison of its antibacterial activity with N-lobe[J]. Systems Microbiology and Biomanufacturing, 2022, 2(2): 345-354.
    [31] WANG Q, FU WL, LU RQ, PAN CL, YI GF, ZHANG X, RAO ZM. Characterization of Bacillus subtilis Ab03 for efficient ammonia nitrogen removal[J]. Systems Microbiology and Biomanufacturing, 2022, 2(3): 580-588.
    [32] ZHU Y, LIU JQ, PARK J, RAI P, ZHAI RG. Subcellular compartmentalization of NAD+ and its role in cancer: a sereNADe of metabolic melodies[J]. Pharmacology & Therapeutics, 2019, 200: 27-41.
    [33] MA T, SHI B, YE ZL, LI XW, LIU M, CHEN Y, XIA J, NIELSEN J, DENG ZX, LIU TG. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene[J]. Metabolic Engineering, 2019, 52: 134-142.
    [34] LV XM, WANG F, ZHOU PP, YE LD, XIE WP, XU HM, YU HW. Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae[J]. Nature Communications, 2016, 7: 12851.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

董雅君,崔世修,刘延峰,李江华,堵国成,吕雪芹,刘龙. 功能膜微域在七烯甲萘醌合成过程中的作用解析[J]. 生物工程学报, 2023, 39(6): 2215-2230

复制
分享
文章指标
  • 点击次数:262
  • 下载次数: 1484
  • HTML阅读次数: 668
  • 引用次数: 0
历史
  • 收稿日期:2023-02-16
  • 录用日期:2023-03-31
  • 在线发布日期: 2023-06-20
  • 出版日期: 2023-06-25
文章二维码
您是第5984901位访问者
生物工程学报 ® 2025 版权所有

通信地址：中国科学院微生物研究所    邮编：100101

电话：010-64807509   E-mail：cjb@im.ac.cn

技术支持：北京勤云科技发展有限公司